Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells

Comparative eye-tracking evaluation of scatterplots and parallel coordinates

We investigate task performance and reading characteristics for scatterplots (Cartesian coordinates) and parallel coordinates. In a controlled eye-tracking study, we asked 24 participants to assess the relative distance of points in multidimensional space, depending on the diagram type (parallel coordinates or a horizontal collection of scatterplots), the number of data dimensions (2, 4, 6, or 8), and the relative distance between points (15%, 20%, or 25%). For a given reference point and two target points, we instructed participants to choose the target point that was closer to the reference point in multidimensional space. We present a visual scanning model that describes different strategies to solve this retrieval task for both diagram types, and propose corresponding hypotheses that we test using task completion time, accuracy, and gaze positions as dependent variables. Our results show that scatterplots outperform parallel coordinates significantly in 2 dimensions, however, the task was solved more quickly and more accurately with parallel coordinates in 8 dimensions. The eye-tracking data further shows significant differences between Cartesian and parallel coordinates, as well as between different numbers of dimensions. For parallel coordinates, there is a clear trend toward shorter fixations and longer saccades with increasing number of dimensions. Using an area-of-interest (AOI) based approach, we identify different reading strategies for each diagram type: For parallel coordinates, the participants’ gaze frequently jumped back and forth between pairs of axes, while axes were rarely focused on when viewing Cartesian coordinates. We further found that participants’ attention is biased: toward the center of the whole plotfor parallel coordinates and skewed to the center/left side for Cartesian coordinates. We anticipate that these results may support the design of more effective visualizations for multidimensional data

Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression

Satb1 and the closely related Satb2 proteins regulate gene expression and higher-order chromatin structure of multigene clusters in vivo. In examining the role of Satb proteins in murine embryonic stem (ES) cells, we find that Satb1−/− cells display an impaired differentiation potential and augmented expression of the pluripotency determinants Nanog, Klf4, and Tbx3. Metastable states of self-renewal and differentiation competence have been attributed to heterogeneity of ES cells in the expression of Nanog. Satb1−/− cultures have a higher proportion of Nanoghigh cells, and an increased potential to reprogram human B lymphocytes in cell fusion experiments. Moreover, Satb1-deficient ES cells show an increased expression of Satb2, and we find that forced Satb2 expression in wild-type ES cells antagonizes differentiation-associated silencing of Nanog and enhances the induction of NANOG in cell fusions with human B lymphocytes. An antagonistic function of Satb1 and Satb2 is also supported by the almost normal differentiation potential of Satb1−/−Satb2−/− ES cells. Taken together with the finding that both Satb1 and Satb2 bind the Nanog locus in vivo, our data suggest that the balance of Satb1 and Satb2 contributes to the plasticity of Nanog expression and ES cell pluripotency

The proteomic signature of recombinant growth hormone in hecreational athletes

Objective: Administration of human growth hormone (hGH) is prohibited in competitive sport and its detection in an athlete's sample triggers an adverse analytical finding. However, the biological processes that are modulated by recombinant hGH are not well characterized and associated blood serum proteins may constitute new biomarkers for hGH misuse. Methods: Thirty-five recreational athletes were enrolled in a study to investigate the time- and dose-dependent response of serum protein levels to recombinant hGH administration. Participants were randomly assigned to 4 groups, receiving 1 of 3 different doses of recombinant hGH or a placebo. Bio samples were collected at 22 time points over a period of 13 weeks, starting 4 weeks before treatment, during 3 weeks of treatment, and at 6 weeks' follow-up. A total of 749 serum samples were analyzed for 1305 protein markers using the SOMAscan proteomics platform. Results: We identified 66 proteins that significantly associated with recombinant hGH administration and dosage, including well known hGH targets, such as IGF1, but also previously unknown hGH-related proteins (eg, protease inhibitors, WFIKKN1, and chemokines, CCL2). Network analysis revealed changes in specific biological pathways, mainly related to the immune system and glucose metabolism. Conclusion: Our analysis suggests that hGH administration affects biological processes more strongly than previously acknowledged. Some of the proteins were dysregulated even after hGH treatment and could potentially be developed into biomarkers for hGH misuse. Moreover, our findings suggest new roles for hGH-associated proteins in the etiology of hGH-related diseases and may indicate new risks that may be associated with hGH misuse.This work is supported by the Biomedical Research Program at Weill Cornell Medicine in Qatar, a program funded by the Qatar Foundation, and the World Anti-Doping Agency. K.S. was also supported by the Qatar National Research Fund (QNRF

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining <i>bona fide</i> NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining <i>bona fide</i> NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology

Sputum Proteome Signatures of Mechanically Ventilated Intensive Care Unit Patients Distinguish Samples with or without Anti-pneumococcal Activity

Mechanically ventilated patients are at risk of contracting pneumonia. Therefore, these patients often receive prophylactic systemic antimicrobial therapy. Intriguingly however, a previous study showed that antimicrobial activity in bronchoalveolar aspirates (here referred to as "sputa") from ventilated patients was only partially explained by antibiotic therapy. Here we report that sputa from these patients presented distinct proteome signatures depending on the presence or absence of antimicrobial activity. Moreover, we show that the same distinction applied to antibodies against Streptococcus pneumoniae, which is a major causative agent of pneumonia. Specifically, the investigated sputa that inhibited growth of S. pneumoniae, while containing subinhibitory levels of the antibiotic cefotaxime, presented elevated levels of proteins implicated in innate immune defenses, including complement and apolipoprotein-associated proteins. In contrast, S. pneumoniae-inhibiting sputa with relatively high cefotaxime concentrations or noninhibiting sputa contained higher levels of proteins involved in inflammatory responses, such as neutrophil elastase-associated proteins. In an immunoproteomics analysis, 18 out of 55 S. pneumoniae antigens tested showed significantly increased levels of IgGs in inhibiting sputa. Hence, proteomics and immunoproteomics revealed elevated levels of antimicrobial host proteins or S. pneumoniae antigen-specific IgGs in pneumococcal growth-inhibiting sputa, thus explaining their anti-pneumococcal activity. IMPORTANCE Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. This suggests the presence of potentially protective antimicrobial agents in their lung environment. Our present study shows for the first time that bronchoalveolar aspirates, "sputa," of ventilated patients in a Dutch intensive care unit were characterized by three distinct groups of proteome abundance signatures that can explain their anti-pneumococcal activity. Importantly, this anti-pneumococcal sputum activity was related either to elevated levels of antimicrobial host proteins or to antibiotics and S. pneumoniae-specific antibodies. Further, the sputum composition of some patients changed over time. Therefore, we conclude that our study may provide a novel tool to measure changes that are indicative of infection-related conditions in the lungs of mechanically ventilated patients